Vascular basement membrane thickening is a prominent and characteristic lesion of diabetic retinopathy. The goal of this project is to test the hypothesis that the thickened vascular basement membrane (BM) occurring in diabetes plays a role in the development and progression of serious structural and functional abnormalities of diabetic retinopathy. The hypothesis is based on recent findings that human retinal vessels manifest alterations that can be induced by the thickened BMs and, in turn, can compromise the integrity of the vessels' inner lining. In retinal capillary cells of diabetic individuals there is increased production of the BM proteins, fibronectin (FN), collagen IV (coil IV), and laminin (LM). Antisense oligonucleotides developed in our laboratory specifically down-regulate overexpression of the three BM components in microvascular endothelial cells grown in high glucose medium. Furthermore, inhibition of FN overexpression with the FN antisense oligonucleotide partially prevented the development of thickened vascular BM in retinal capillaries of galactose-fed rats, an animal model of diabetic retinopathy, with beneficial consequences to histological lesions. To completely prevent or reverse vascular BM thickening, and regulate functional abnormalities, the development of a more effective antisense strategy is necessary. Having identified antisense oligos that independently down-regulate FN, coil IV or LM expression in retinal vascular cells, in the proposed studies we plan to: (1) Establish whether combined antisense oligo approach prevents vascular BM thickening in rat retinas, and affects FN, coil IV, LM turnover in matrix. (2) Determine if downregulation of the specific BM genes reverses BM thickening, reduces vascular lesions, and affects cellular processes in retinas of diabetic rats. (3) Determine whether high glucose-induced or diabetes-induced altered expression of BM components plays a role in vascular permeability. FN, coil IV, and LM protein level will be monitored by Western blot analysis and immunohistochemistry; RNA level will be analyzed by RT-PCR. Retinal capillary BM width will be measured by morphometric analysis of electron micrographs. Vascular lesions will be assessed from retinal trypsin digests and image analysis. Findings from this project will establish or exclude a pathogenetic link between a discrete biosynthetic abnormality and the development of structural and functional lesions of diabetic retinopathy.